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The Electrical Properties of Bufo Marinus Na+, K+-Atpase A

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The Electrical Properties of Bufo Marinus Na+, K+-Atpase A The Electrical Properties of Bufo marinus Na+, K+-ATPase A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Jingping Hao November 2009 © 2009 Jinging Hao. All Rights Reserved. 2 This dissertation titled The Electrical Properties of Bufo marinus Na+, K+-ATPase by JINGPING HAO has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Ralph A. DiCaprio Professor of Biological Sciences Benjamin M. Ogles Dean, College of Arts and Sciences 3 ABSTRACT HAO, JINGPING, Ph.D., November 2009, Biological Sciences The Electrical Properties of Bufo marinus Na+, K+-ATPase (145 pp.) Director of Dissertation: Ralph A. DiCaprio The Na+, K+-ATPase is a primary active transporter that transports three Na+ out of the cell and two K+ into the cell. The pump produces a net charge transfer, and is therefore electrogenic, so the generated current can serve as a measure of the pump activity. In order to test the current-voltage (I-V) relationship of the sodium pump, Bufo marinus Na+, K+-ATPase alpha and beta subunits were cloned and their functional properties were tested using a Xenopus laevis expression system and two-electrode voltage clamp techniques. The steady state pump current is known to be voltage dependent (Sagar and Rakowski, 1994), and can be activated by extracellular K+, and inhibited by ouabain, which is a specific inhibitor of the sodium pump. We determined the ouabain-sensitive current as a measure of the pump-mediated current with different concentrations of ouabain used to inhibit the exogenous K+ activated pump current. The voltage dependence of the ouabain-sensitive pump current was shown to be due to the voltage dependence of the sodium pump turnover rate, because the inhibition of pump current by ouabain did not depend on membrane potential. The Ki value for ouabain inhibition is 30 ± 3 µM. Pump current activated by addition of K+ was not equal to the current inhibited by ouabain, contrary to results previously obtained that measured the endogenous Xenopus pump current using the compound tetraethylammonium (TEA+). TEA+ has been widely used to block K+ channels and permit measurement of pump 4 current simply by addition of external K+. A series of experiments was perfomed in the absence of external K+ to test if TEA+ is itself capable of activating Bufo pump current. In both high (100 mM) and Na+-free external conditions, TEA+ elicited K+-like activation of the ouabain sensitive current. TEA+ activates an electrogenic pump current with a lower affinity than that of K+. The X-ray crystal structure of pig renal Na+, K+ -ATPase at 3.5 Å resolution has been determined with two occluded rubidium ions. These two sites are expected to be occupied by Na+ and K+ alternatively. The position of the third Na+ binding site is still hypothetical. Mutations were used to examine the effects of changing the candidate amino acids of the binding site. These mutations change the voltage dependence and apparent affinity of extracellular Na+ binding. These results suggest that the third Na+ binding site is located between the fifth, sixth, and ninth transmembrane helices of the Na+, K+ -ATPase alpha subunit. Approved: _____________________________________________________________ Ralph A. DiCaprio Professor of Biological Sciences 5 DEDICATION Dedicated to my late advisor Robert F. Rakowski 6 ACKNOWLEDGMENTS I would like to express my gratitude to all those who gave me the ability to complete this dissertation. My deepest gratitude is to my late advisor, Dr. Robert Rakowski. I have been amazingly fortunate to be one of his students. He gave me the freedom to explore on my own, and at the same time gave me guidance when I had difficulties. He taught me how to question thoughts and express ideas. His support and patience helped me overcome many obstacles. I would like to thank my current advisor, Dr. Ralph DiCaprio. Without his encouragement and advice, I would not have finished my dissertation. I would also like to thank my committee members, Dr. Janet Duerr, Dr. Michael Rowe, Dr. Soichi Tanda, and Dr. Jennifer Hines for their help in this project. I would like to thank my lab mate and friend, Yanli Ding. She gave me much help and support not only in my research but also in my life. Finally, I sincerely thank my parents, grandparents, and my husband, Jiong Hu. They always believed in me and directed their efforts to help me get through these years. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments............................................................................................................... 6 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Chapter 1: Introduction ..................................................................................................... 14 I. Structure of the sodium pump .................................................................................. 14 II. Function of the sodium pump: Post-Albers Transport Cycle of Na+, K+-ATPase ... 18 III. Sodium binding sites ............................................................................................... 21 IV. Specific pump inhibitor: Ouabain ........................................................................... 30 V. The external access channel model .......................................................................... 34 VI. Electrical properties of ion transport ...................................................................... 35 VII. K+ channel blocker: Tetraethylammonium ........................................................... 38 Chapter 2 Materials and methods ..................................................................................... 40 I. Experimental Solutions ............................................................................................. 40 II. Preparation of cRNA ................................................................................................ 40 III. Production of single-point mutations ...................................................................... 41 IV. Isolation and maintenance of Xenopus oocytes ...................................................... 41 V. Injection of oocytes .................................................................................................. 42 VI. Immunofluorescent staining ................................................................................... 42 8 VII. Electrophysiological measurements ...................................................................... 43 VIII. Measurement of the pump I-V relationship ......................................................... 45 A. Basic Mechanism .............................................................................................. 45 B. Experimental protocol for the measurement of steady state pump current ...... 49 C. Control experiment ........................................................................................... 50 D. Unsubtracted I-V curves ................................................................................... 51 E. Difference current ............................................................................................. 52 Chapter 3 Results .............................................................................................................. 54 I. Inhibition of pump current by ouabain does not depend on membrane potential ..... 54 II. Electrogenic transport of tetraethylammonium (TEA+) by Bufo marinus Na+, K+- ATPase .......................................................................................................................... 60 Part 1. TEA+ activated current with and without external K+ ................................... 60 Part 2. Ouabain inhibited current in TEA+-free condition ........................................ 72 Part 3. pH effect on the TEA+ activated pump current ............................................. 77 Part 4. Electrical properties of TEA+ activation ....................................................... 83 III. The third Na+ binding site ..................................................................................... 106 A. Mutation studies .............................................................................................. 106 B. Detecting the protein at the oocyte surface by using immunofluorescent staining .................................................................................................................... 115 Chapter 4 Discussion ...................................................................................................... 118 I. Ouabain binding to the Na+, K+-ATPase is voltage independent ............................ 118 II. pH effect on the TEA+ activated pump current ...................................................... 120 9 III. Voltage dependent TEA+ binding on the Na+, K+-ATPase ................................. 122 1. Access Channel Model ......................................................................................
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